Portal de Periódicos da CAPES

Direct imaging of lateral movements of AMPA receptors inside synapses

2003; Springer Nature; Volume: 22; Issue: 18 Linguagem: Inglês

10.1093/emboj/cdg463

1460-2075

Catherine Tardin,

Neural dynamics and brain function

Article15 September 2003free access Direct imaging of lateral movements of AMPA receptors inside synapses Catherine Tardin Catherine Tardin Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Laurent Cognet Laurent Cognet Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Search for more papers by this author Cécile Bats Cécile Bats Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Brahim Lounis Brahim Lounis Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Search for more papers by this author Daniel Choquet Corresponding Author Daniel Choquet Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Catherine Tardin Catherine Tardin Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Laurent Cognet Laurent Cognet Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Search for more papers by this author Cécile Bats Cécile Bats Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Brahim Lounis Brahim Lounis Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France Search for more papers by this author Daniel Choquet Corresponding Author Daniel Choquet Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France Search for more papers by this author Author Information Catherine Tardin1,2, Laurent Cognet1, Cécile Bats2, Brahim Lounis1 and Daniel Choquet 2 1Centre de Physique Moléculaire Optique et Hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France 2Laboratoire de Physiologie Cellulaire de la Synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint-Saëns, 33077 Bordeaux, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4656-4665https://doi.org/10.1093/emboj/cdg463 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single- molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses. Introduction AMPA glutamate receptors (AMPARs) are ligand- activated cation channels concentrated in the postsynaptic density (PSD) that mediate fast excitatory neurotransmission in the CNS (Dingledine et al., 1999). Concentration of AMPARs at PSDs is thought to result from their stabilization by interactions with specific intracellular scaffolding proteins and cytoskeletal elements (Braithwaite et al., 2000; Nusser, 2000; Scannevin and Huganir, 2000). AMPARs constitutively cycle in and out of the postsynaptic membrane at a rapid rate through endo- and exocytosis. The regulation of the balance between these processes may account for the rapid variations in receptor composition of the PSD during synaptic plasticity (reviewed in Carroll et al., 2001; Malinow and Malenka, 2002; Sheng and Kim, 2002). A simplified scheme would be that postsynaptic LTP involves increased exocytosis of AMPARs, while LTD would be underlied by increased endocytosis of receptors. Recently, a role for receptor diffusion within the plasma membrane has been suggested in AMPARs trafficking in and out of PSDs. First, GluR2 containing AMPARs diffuse rapidly in the extra-synaptic membrane and stop reversibly in the periphery of synapses (Borgdorff and Choquet, 2002). Secondly, endo- or exocytosis may occur at the periphery of synapses rather than directly at PSDs. For endocytosis, clathrin assembly occurs at hotspots laterally to the PSDs (Blanpied et al., 2002). LTD or glutamate application trigger an increase in the amount of endocytosed AMPARs (Carroll et al., 1999a,b; Beattie et al., 2000; Wang and Linden, 2000). Endocytosis of AMPARs may first require their dispersal from the synaptic to the extrasynaptic membrane through lateral diffusion, as glutamate by itself does not increase the efficiency of the endocytotic pathway (Zhou et al., 2001). Reciprocally, whether there are also hotspots for exocytosis of receptors outside of the PSD is unknown. At early times after exocytosis, new GluR1 subunits are diffusively distributed along dendrites. This is followed by their lateral translocation and accumulation into synapses (Passafaro et al., 2001). Delivery of receptors to the PSD after exocytosis may thus also involve diffusion. Mechanisms involved in the regulation of the accumulation of AMPARs at synaptic sites through lateral diffusion are emerging. Local increases in intracellular calcium drastically reduce AMPAR diffusion rate (Borgdorff and Choquet, 2002). Stargazin, a protein that links AMPARs to PSD-95, might regulate AMPAR trafficking between the synaptic and extra-synaptic membrane (Schnell et al., 2002). Therefore, it is likely that diffusion of receptors in the plane of the membrane is necessary for their removal or addition to and from the PSD, although this has never been visualized directly. If receptors enter and leave synapses through lateral diffusion, they have to unbind from the postsynaptic scaffold, diffuse through the PSD and exit the synapse. Clusters of receptors mimicking the PSD can be induced in the synaptic and extra-synaptic membrane by co-expression of scaffold and receptor molecules. At the extra-synaptic clusters, we had directly visualized entry and exit of receptors from the clusters at high rates using single particle tracking (Meier et al., 2001; Sergé et al., 2002). However, the size of the particle (500 nm) precluded tracking of receptors inside the synaptic cleft. In this study, we use the single-molecule fluorescence imaging approach (e.g. Schmidt et al., 1995; Dickson et al., 1996; reviewed in Moerner and Orrit, 1999; Weiss, 1999) to localize and track GluR2-containing AMPA receptors inside synaptic sites below the optical diffraction limit. We show that a large proportion of AMPARs diffuse inside synapses and that this diffusion is regulated during protocols that modify receptor accumulation at synapses. We propose that receptor disappearance from postsynaptic sites involve their dispersal through increased lateral diffusion while receptor accumulation involves their delayed stabilization after diffusion. Results Single molecule imaging in live neurons Anti-GluR2 antibodies were labeled with Cy5 or Alexa-647 molecules at low labeling ratio (mean labeling ratio of 0.4 dye per antibody) so that individual antibodies were labeled at most with one fluorophore. A small proportion of surface expressed AMPA receptors containing the GluR2 subunit were selectively labeled in live neurons through short incubations with these antibodies. We could thus image and resolve discrete fluorescence spots with an epifluorescence imaging setup (Schmidt et al., 1995; Dickson et al., 1996). The majority of the fluorescence spots (75 ± 6%, n = 80 neurons) exhibit one-step photobleaching (Figure 1E; Supplementary movies 1–4, available at The EMBO Journal Online) and not a gradual decay as for ensemble photobleaching. The width of these spots corresponds to the point-spread function of the microscope and the signal originating from them ranges from 500 to 1000 counts per 30 ms. Thus, these fluorescence spots have all the hallmarks of individual fluorescent molecules (see Supplementary figure 1; reviewed in Moerner and Orrit, 1999; Weiss, 1999) bound to GluR2 receptors. Only these spots were thus retained for analysis. The imaged single molecules were optically well resolved (Figure 1C; Supplementary movies 1–4) and their density on the cell surface was much less than 1/μm2. This indicates that antibody incubation did not result in cross-linking of more than two GluR2-containing AMPARs, the anti-GluR2 being bivalent. This was further supported by immunocytochemistry experiments: the apparent level of receptor clustering was smaller when incubation with anti-GluR2 was performed on live compared with fixed cells (percentage of clustered receptors 15 ± 7%, n = 12, and 23 ± 9%, n = 12, respectively). However, this does not rule out the possibility that single molecule tracking follows the movement of a natural cluster of receptors, only one receptor being labeled. Figure 1.Single-molecule fluorescence detection of GluR2-containing AMPARs. (A–C) Simultaneous images of a neurite of a living neuron as seen by differential interference contrast (A) and epifluorescence of FM1-43 on a green channel and Cy5 on a red channel (B, C). (B) Synaptic sites stained by depolarization-induced uptake of FM1-43. (C) Diffraction limited spot image of a single Cy5-anti-GluR2 antibody. The molecule is co-localized with one of the three synaptic sites of (B) (see also Supplementary movies). Scale bar = 1 μm. (D) Three-dimensional representation (intensity in the vertical axis) of the fluorescence of the molecule in (B). Scale bar is counts per 30 ms. (E) Recording of the fluorescence intensity of a single Cy5-anti-GluR2 molecule over time displaying the characteristic one-step photobleaching. (F) Double staining of surface GluR2 on a live neuron (left and red in the merge) and presynaptic terminals with an anti-vesicular glutamate transporter (middle and green in the merge). (G) Surface proportion of the detected AMPARs S(r) (see text) for single molecules recorded in the presence of TTX. AMPARs accumulate at and close to synaptic staining maxima. Download figure Download PowerPoint Trajectories of such molecules were reconstructed from image series recorded at a rate of 33 Hz (see movies in Supplementary data). The length of the trajectories varied from 0.1 to 0.5 s up to 4 s, depending on the photobleaching time of the molecule (mean ± SD 244 ± 318 ms, n = 3078 molecules). The mean-square displacement (MSD) corresponding to trajectories of individual fluorescent molecules dried on glass shows that individual molecules are pointed within 45 ± 5 nm accuracy (Schmidt et al., 1995; Thompson et al., 2002) (Figure 2B, trajectory 1). Figure 2.(A and B) Illustrative examples of AMPARs movements. (A) Examples of trajectories of individual molecules. Cy5-anti-GluR2 fixed on a coverslip (1). The other trajectories correspond to single Cy5-anti-GluR2 bound to AMPARs in living dendrites. The trajectories recorded in synaptic regions (see text) are indicated in green. The trajectories recorded in extra-synaptic domains are indicated in red. Trajectories 2 and 3 stayed within synaptic sites; trajectory 4 evolved entirely in the extra-synaptic membrane; trajectory 5 started in an extra-synaptic region and then entered a synaptic site. (B) Plots of the MSD versus time intervals τ for four trajectories shown in (A). Trajectories 2 and 3, which were both synaptic, were less mobile than trajectory 1, but differed: trajectory 2 corresponds to a slowly mobile receptor in a confined area, 3 to an immobile receptor. (C–F) Statistical analysis of AMPAR movements. (C and D) Differential mobility of synaptic and extra-synaptic AMPARs measured at 37°C in the presence of TTX. (C) Histograms of the instantaneous diffusion constant for 306 AMPARs trajectories detected in extra-synaptic regions of >20 neurons. (D) Same histograms for 187 AMPARs trajectories detected in synaptic sites of >20 neurons. Binning of the major histograms is 0.075 μm2/s. The insets correspond to the same data with a binning of 0.007 μm2/s. (E and F) Single-molecule analysis of AMPARs diffusion: plots of the mean MSD and derived from the analysis of the square displacements for τ = n × 30 ms (n = 1–11). (E) is linear with time and reveals free diffusion for fast diffusing extra- synaptic AMPARs. (F) are undistinguishable for synaptically located (filled circle) and slowly diffusing extra-synaptic (open squares) trajectories and reveal confined movement. Download figure Download PowerPoint GluR2 molecules are imaged in synapses We first analyzed the spatial distribution of AMPARs with respect to synaptic sites in bulk immunocytochemistry experiments and at the single molecule level in live neurons. For both types of experiments, live neurons were incubated for short periods with anti-GluR2 antibodies (10 min). For bulk visualization of receptors only, this step was followed by fixation and amplification of the signal through secondary antibodies. In immunocytochemistry experiments, AMPARs accumulated in front of glutamatergic presynaptic terminals specifically stained by the vesicular glutamatergic transporter BNPI/VGLUT1 (Figure 1F). A similar accumulation was previously observed using other presynaptic markers (Carroll et al., 1999b; Noel et al., 1999). In live neurons, presynaptic terminals were stained with FM1-43 or rhodamine 123 (Figure 1B; Supplementary movies 2–4). We measured the distance r between each individual AMPARs and the center of the closest stained synaptic site. We plotted S(r), the proportion of individual molecules per unit surface, as a function of r (Figure 1G). Individual AMPARs are strongly enriched (∼10 times) at and close to (<300–400 nm) synaptic sites. Altogether, these experiments indicate that synaptic AMPARs can be stained through incubation with antibodies in live neurons. They further establish that live staining of presynaptic terminals is a valid approach to discriminate between synaptic and extra-synaptic regions and analyze AMPAR movements in these membrane domains. Diffusion characteristics are correlated with localization with respect to synapses: examples Single molecule trajectories were initially sorted into two main categories: the first one corresponds to molecules located at a distance <300 nm from the center of a presynaptic staining and are referred to as 'synaptic' throughout this work using this cut-off criterion (see Materials and methods). Three of these trajectories are illustrated by examples 2 and 3 in Figure 2A, and in Supplementary movie 4. The second group category corresponds to all the others, including molecules found in the periphery of synapses. They are referred to as 'extra-synaptic' and illustrated by example 4 in Figure 2A, and in Supplementary movies. We observed a variety of mobility behaviors for the receptors, which are correlated with the localization. They ranged from highly mobile receptors only seen in extra-synaptic regions (exemplified by trajectory 4 in Figure 2A) to mildly mobile (trajectory 3) or immobile (trajectory 2). The latter two were mainly found at synaptic sites. Strikingly, individual receptors directly entering and leaving synaptic domains could be observed on occasion (∼1–2% of all trajectories; e.g. trajectory 5 in Figure 2A, and Supplementary movie 2). The MSD is widely used to extract diffusion characteristics from trajectories. In Figure 2B, the MSD is plotted as a function of time for trajectories 1–4 illustrated in Figure 2A. The extra-synaptic mobile receptor diffused freely, as indicated by its linear MSD, while the movement of mobile receptors in synapses was confined to a domain, as indicated by the plateau reached by its MSD over time. The domain size was typical for a synapse (∼400 nm; see below). Diffusion characteristics are correlated with localization with respect to synapses: statistical and analytical analysis We performed a statistical analysis on the mobility of AMPARs in each region (synaptic or extra-synaptic). We first analyzed the trajectories of AMPARs in the presence of TTX to block spontaneous neuronal activity (the examples shown in Figure 2A and B were part of 493 trajectories from more than 20 different neurons recorded at 37°C). After sorting the 493 trajectories with respect to their localizations in the two regions, we calculated the instantaneous diffusion coefficient, D, for each trajectory, from linear fits of the first three to five points (corresponding to 90–150 ms) of the MSD (Kusumi et al., 1993; Sergé et al., 2002) using: MSD (τ) = < r2 > (τ) = 4Dτ. Distributions of D for synaptic and extra-synaptic receptors were strikingly different (Figure 2C and D), synaptic receptors diffusing more slowly than extra-synaptic ones. In the extra-synaptic region, we distinguished three populations from the histograms (Figure 2C). For the majority (66%) of the molecules, the diffusion coefficient was in the range of 0.1–1 μm2/s (mean ± SEM 0.45 ± 0.05 μm2/s, n = 202 trajectories, seven experiments) in good agreement with measurements made by single particle tracking (Borgdorff and Choquet, 2002). The two other populations correspond to molecules which were either immobile (8%, D <7 × 10−3 μm2/s, n = 25) or diffused slowly (26% with D <0.1 μm2/s, mean 0.05 ± 0.01 μm2/s, n = 82; see inset in Figure 2C). In stained synaptic sites, receptors could be separated into two populations (Figure 2D). Half of the receptors were immobile, whereas the other half diffused with a diffusion coefficient between 1.5 × 10−2 and 0.1 μm2/s (mean 0.054 ± 0.005 μm2/s, n = 100). At a given location (synaptic or extra-synaptic), AMPARs with different mobilities were found. In particular, both mobile and immobile receptors were observed successively at the same synaptic site within the same recording sequence in 21 cases, showing that the two receptor's behaviors do not arise from receptors present in separate types of synapses. Moreover, these observations show that on the time scale of our experiments, the movement of synaptic AMPARs is not that of the whole PSDs. In order to analytically characterize the diffusion properties of each subpopulation at each location, we used a second approach based on the distribution of the squared displacements of the molecules (Schuetz et al., 1997). This approach allows us to unravel and analyze multiple diffusion types in each compartment without having to classify the MSDs of the individual molecules, thus avoiding possible bias by an arbitrary sorting. As a result, three categories of receptor movements were also found by this analytical analysis (see Supplementary figure 2), characterized by the time dependence of their mean MSD, , where i = 0–2. Fast mobile receptors (i = 2) were exclusively found in extra-synaptic regions, while the slowly mobile (i = 1) and immobile (i = 0) receptors were mainly found in synaptic domains (Figure 2E and F; Supplementary data). Thus, two independent analysis protocols, i.e. distributions of individual D values (Figure 2C and D) and distributions of squared displacements (Supplementary figure 2; Figure 2E and F), establish the existence of different receptor populations in terms of mobility. Moreover, the latter analysis paradigm establishes the type of diffusion for the two mobile populations. On one hand, is linear with time, indicating that these extra-synaptic receptors undergo free Brownian diffusion (at least up to 330 ms; Figure 2E) with a mean diffusion constant of 0.37 ± 0.04 μm2/s. On the other hand, saturates with time (Figure 2F), a signature of spatially restricted diffusion (Kusumi et al., 1993; Sergé et al., 2002). These were found, with very similar properties, in both the synaptic and extra-synaptic receptor populations, although in different proportions (45 ± 5% for synaptic and 25 ± 5% for extra-synaptic receptors; data not shown). The diffusion coefficient D1 and the diameter L of the domain within which diffusion is restricted can be derived (Kusumi et al., 1993) from: Least-square fitting of the data by Equation 1 gives D1 = 6 ± 2 × 10−2 μm2/s and a domain size L = 300 ± 20 nm. The domain size is in good quantitative agreement with the synaptic sizes given by electron microscopy (Schikorski and Stevens, 1997; Takumi et al., 1999), suggesting that mobile receptors explore the whole postsynaptic domain. Receptors that we labeled as extra-synaptic and which nevertheless displayed restricted diffusion could in fact pertain to unstained synapses. Alternatively, we have previously shown that extra-synaptic receptors aggregated by scaffolding proteins display a similarly restricted mobility (Meier et al., 2001; Sergé et al., 2002). Contribution of endocytosis to receptor mobility GluR2 containing AMPARs are known to undergo continuous endo/exocytosis and recycling (Carroll et al., 2001). Our experiments did not detect newly exocytosed receptors as no free antibody was present during the recordings. In contrast, antibodies are known to be endocytosed together with the receptors to which they are bound (e.g. Luscher et al., 1999). We first measured by imunohistochemistry on live cells the global level of endocytosis of antibody-tagged receptors. Neurons were incubated 10 min with anti-GluR2 at 37°C, washed, and further incubated 15 min at 37°C before being fixed and stained for surface and endocytosed receptors (see Materials and methods). We found that 24 ± 5% (mean ± SEM, 11 neurons) of the receptors were endocytosed in neurites. Then, to investigate specifically the mobility of endocytosed receptors, we first incubated labeled cells for 30 min at 37°C to allow endocytosis to occur, then removed surface labeling by acid wash prior to performing single molecule experiments. Surface staining was decreased by 80 ± 9% (n = 10 neurons) by this treatment. The mobility of the internalised receptors is shown Figure 3. We found that after acid wash, 79% of the total receptors (synaptic plus extra-synaptic) were immobile, compared with 25% in control recordings. Figure 3.(A and B) Histograms of the cumulative distribution of instantaneous diffusion constant of synaptic and extra-synaptic receptors in control conditions (A) and after acid wash to detect specifically endocytosed AMPARs (493 and 975 trajectories, respectively). Binning as in Figure 2C. (C and D) Temperature dependence of AMPAR diffusion. (C) Mean instantaneous diffusion constant for freely diffusing AMPARs in extra-synaptic regions (hollow) and for diffusing AMPARs (with restricted diffusion) at synaptic sites (filled) as a function of TI and TE, the incubation temperatures during the labeling by anti-GluR2, TI, or during the experiments, TE. (D) Fraction of immobile over mobile receptors in synaptic sites for different temperatures. Download figure Download PowerPoint To further investigate the contribution of receptor endocytosis to the proportion of immobile receptors, we performed temperature block of endo/exocytosis in the presence of TTX. First, in immunohistochemistry experiments, we found that the percentage of endocytosis after 15 min at 20°C dropped to 10 ± 4% (eight neurons). Secondly, 433 (respectively 422) single-molecule trajectories from 20 different live neurons were recorded at 37°C (respectively 20°C) after antibody incubation at 20°C to reduce the amount of internalized receptors. The mean diffusion constants of the mobile population of receptors in synaptic sites was not different between 37 and 20°C (Figure 3C); however, it decreased by a factor of three at 20°C in the extra-synaptic regions (including all mobile extra-synaptic receptors it varied from 0.32 ± 0.04 μm2/s, n = 207 at 37°C to 0.11 ± 0.04 μm2/s, n = 82 at 20°C). This shows that diffusion of receptors at synaptic sites is not limited by the viscosity of the membrane, as it is likely to be in extra-synaptic regions. This decrease in diffusion constants in extra-synaptic regions led to an apparent 2-fold increase in the proportion of receptors counted as immobile (D 40% when going from 37 to 20°C (see Figure 3D). This decrease is larger than what could be expected from a simple block of endocytosis and may arise from additional phenomena. In any case, this confirms that endocytosed receptors belong to the immobile population. All data presented below were obtained from recordings performed at 37°C after antibody incubations at 20°C. Regulation of AMPARs mobility inside synapses Postsynaptic plasticity of glutamatergic synapses is mediated in large part by the regulation of AMPAR trafficking (reviewed in Carroll et al., 2001; Malinow and Malenka, 2002; Sheng and Kim, 2002). Protocols that induce plasticity of synaptic transmission in culture result in changes of AMPAR concentration at synapses and are thought to mimic at the molecular level the processes of LTP and LTD (Carroll et al., 1999a; Beattie et al., 2000; Lin et al., 2000; Lu et al., 2001; Passafaro et al., 2001). Changes in AMPAR numbers at synapses have mainly been attributed to changes in endocytosis or exocytosis of receptors. These membrane traffic events are likely to occur outside PSDs (Passafaro et al., 2001; Blanpied et al., 2002), which implies that receptor diffusion in the plane of the plasma membrane should participate to the changes in synaptic receptor numbers. Here, we analyze whether AMPARs diffusion inside and outside synapses is regulated by glutamate application and changes in synaptic activity. We used bath application of glutamate to decrease the number of surface-expressed AMPARs (Carroll et al., 1999a; Beattie et al., 2000) through mechanisms that may be shared by LTD (Carroll et al., 1999b; Man et al., 2000). This protocol is referred herein as 'Glut'. Conversely, we also used activation of synaptic release of glutamate through blocking inhibitory neurotransmission by biccuculine and strychnine, together with potentiation of NMDARs by glycine to increase the number of surface AMPARs through mechanisms that may be similar to those of LTP (Lu et al., 2001; Passafaro et al., 2001). This protocol is referred herein as 'Bic/Gly'. Our control condition was, as previously, recordings in the presence of TTX. As a further resting condition, we also used intracellular BAPTA to chelate pre- and postsynaptic intracellular calcium. We first verified by immunohistochemistry on live neurons (data not shown) that these protocols induced reciprocal changes in surface expression of AMPARs (Carroll et al., 1999a; Beattie et al., 2000; Lin et al., 2000; Lu et al., 2001; Passafaro et al., 2001). Indeed, bath application of 100 μM glutamate induced a 85% increase in the percentage of endocytosed AMPARs within 15 min. This corresponded to a loss of 22% of the surface receptors. Bath application for 5 min of 20 μM biccuculine and 1 μM strychnine together with 200 μM glycine induced a 59% increase (SD 19%, n = 10) in surface AMPARs. We then studied the effect of these protocols on receptor mobility at the single molecule level. In a first series of experiments, we labeled surface receptors at 20°C for 10 min and applied the different pharmacological agents during the recordings (Figure 4A). The diffusion of extra-synaptic receptors was not significantly modified by these treatments (Figure 4B). In contrast, bath glutamate induced a strong (55%) increase of the mean diffusion constant of AMPARs inside synapses (Figure 4C). This was accompanied by a 30% reduction in the proportion of immobile synaptic receptors (Figure 4D). This is surprising, since glutamate promotes AMPARs endocytosis and endocytosed receptors are mostly immobile (Figure 3B). A tempting explanation for this discrepancy is that glutamate treatment induces the accumulation of endocytotic vesicles mainly in the cell body rather than in neuri